The present invention relates to a plasmid which can be used, in particular, for transferring a heterologous gene into, and expressing it in, a bacterium, in particular a lactic acid bacterium.
The plasmids of the lactic acid bacteria can be divided into two groups depending on their mode of replication, i.e. either a mode of replication in accordance with the rolling circle (RCR) model or a mode of replication which is similar to that of the chromosome (Theta). Genetic elements which are obligatory or otherwise, depending on the mode of replication of the plasmid, are present in the nucleic acid sequence of the plasmid. These genetic elements can be genes which are directly involved in the replication of the plasmid, specific nucleic acid sequences and nucleic acid sequences which form specific structures.
Up until now, many cloning and expression vectors which have been developed for the lactic acid bacteria have been based on RCR plasmids which have been isolated from lactic acid bacteria (Leenhouts et al., 1991). While their main advantage resides in their broad host spectrum, their unstable nature represents a barrier to using them.
Theta plasmids derived from lactic acid bacteria have been described (Hayes et al., 1990; Kiewiet et al., 1993, Frxc3xa8re et al., 1993). In particular, they display good stability and provide the possibility of transferring inserts of substantial size. Their main disadvantage is, however, that of a narrow host spectrum. For this reason, broad-host-spectrum vectors of the theta type used for cloning and expressing heterologous genes in lactic acid bacteria have been constructed from plasmids which have been isolated from enterococci (Simon and Chopin, 1988) or staphylococci (Le Chatelier et al., 1993). However, it is difficult to envisage using them for food-processing purposes in particular because they are not harmless.
The authors of the present invention have now isolated a plasmid which has been given the reference pTXL1, which is harboured by lactic acid bacterial strains, i.e. the strains Leuconostoc mesenteroides Y110 and Leuconostoc mesenteroides Y111, respectively, whose mode of replication has been established as not being that of the RCR type and which exhibits a high degree of stability and a broad host spectrum within lactic acid bacteria.
Plasmids which possess these properties are novel and are described for the first time within the context of the present invention.
The invention therefore relates to a plasmid whose mode of replication is not of the RCR type and which is capable of being transferred stably within lactic acid bacteria which belong to at least three different genera.
Advantageously, the plasmid is derived from lactic acid bacteria, in particular lactic acid bacteria of the genus Leuconostoc, in particular Leuconostoc mesenteroides. 
Within the meaning of the present invention, the phrase plasmid which is capable of being transferred stably within Leuconostoc mesenteroides lactic acid bacteria is understood as signifying that the plasmid is maintained in the host cell into which it has been transferred, in the absence of selection pressure, after 75 generations, preferably after 90 generations, and more especially after 100 generations, with an apparent size which is equal to the size of the native plasmid apart from any inserts which it may possibly contain, including when it has been modified by the insertion of a sequence of a size as great as 4 kb, as long as the insertion is not effected into the parts of the plasmid which are required for its replication.
The plasmids according to the invention can advantageously be stably transferred into lactic acid bacteria which belong to the genera Leuconostoc; Lactobacillus, Pediococcus and Enterococcus, in particular the following species Leuconostoc mesenteroides, Lactobacillus sake and Pediococcus acidilactici. 
The inventors, who are the authors of the present invention, have furthermore sequenced the plasmid pTXL1 and analysed its nucleic acid sequence.
They have located and determined a sequence fragment which is designated SEQ ID No. 1 below, which has a length of 1346 bp, and which contains all the elements which are required for the plasmid to replicate autonomously. This sequence has been designated xe2x80x9cminimum repliconxe2x80x9d.
The invention therefore also relates to a plasmid as previously defined, which plasmid comprises the nucleotide sequence SEQ ID No. 1 or a sequence which differs from this sequence by the insertion, deletion or mutation of from one to several base pairs, and which retains the ability to replicate.
The total nucleic acid sequence of plasmid pTXL1 has been determined and is designated sequence SEQ ID No. 2.
The invention therefore also relates to a plasmid as previously defined, which plasmid comprises the nucleotide sequence SEQ ID No. 2 or a sequence which differs from this sequence by the insertion, deletion or mutation of one or several base pairs and which retains the ability of the plasmid to replicate stably in bacterial host cells which are of the lactic acid type and which belong to at least three different genera.
Two different strains of lactic acid bacteria harbouring the plasmid have been deposited in a microorganism collection.
The strain Y110 was thus deposited in the Collection Nationale de Culture de Microorganismes (CNCM) [National Collection of Microortanism Cultures] (Institut Pasteur, 28 rue du Docteur Roux, 75724, Paris cedex 15, France) on Oct. 30, 1997 under the number I-1936.
The strain Y111 was thus deposited in the Collection Nationale de Culture de Microorganismes (CNCM) on Oct. 30, 1997 under the number I-1937.
The plasmids, comprising, where appropriate, a heterologous sequence inserted into the plasmid DNA, are transferred into the host cells using known techniques.
Mention may, in particular, be made of the technique of electroporation, in particular that electroporation technique developed by transforming lactic acid bacteria, in particular Leuconostoc, Lactobacillus and Pediococcus.
The invention also relates to bacterial host cells which harbour a plasmid according to the invention, in particular the strains Leuconostoc mesenteroides Y110 and Y111 harbouring the plasmid pTXL1.
Because of the breadth of the host spectrum, the plasmids according to the invention constitute outstanding tools for cloning and expressing heterologous nucleotide sequences in host lactic acid bacteria.
In particular, the plasmids according to the invention can be used for expressing heterologous proteins, such as nisin and mesentericin Y105, which are bacteriocins, proteins for resistance to these bacteriocins, also termed immunity proteins, and/or a protein for resistance to an antibiotic, for example erythromycin, in host cells, in particular lactic acid bacteria.
The expression of heterologous proteins of the abovementioned type is particularly attractive inasmuch as it makes it possible, when the heterologous protein is a bacteriocin, for a lactic acid bacterium, which is present in a medium for a given function and which is totally harmless, to be caused to secrete a substance which is capable of preventing the growth of other bacteria which are undesirable if not to say pathogenic, in particular Listeria bacteria, which have a tendency to grow in lactic acid media.
When the heterologous protein is the protein for immunity to a bacteriocin, the expression of this protein has the value of conferring on the host lactic acid bacterium resistance to the bacteriocin while at the same time preventing the growth of unwanted harmful or pathogenic bacteria.
Similarly, when the heterologous protein is a protein for resistance to an antibiotic, it is then possible to add the antibiotic in question to the medium in which the host bacterium is present without harming the growth and metabolism of this host bacterium while at the same time acting on the growth of unwanted microorganisms.
The invention therefore also relates to the use of a plasmid as described above as a tool for cloning and expressing heterologous nucleic acid sequences in host cells, in particular lactic acid bacteria, in particular the lactic acid bacteria which are mentioned above.
The invention thus also relates to a modified plasmid which is obtained from a plasmid as previously described by inserting a heterologous nucleic acid sequence, preferably a nucleic acid sequence which encodes a heterologous protein or peptide.
The nucleic acid sequence of interest can be inserted, using the appropriate molecular biological tools, in particular restriction enzymes, ligase, etc., into any site in the nucleic acid sequence of the plasmid, in particular in the sequence SEQ ID No. 2 apart from the sequence SEQ ID No. 1.
The length of the nucleic acid sequence encoding the heterologous protein or peptide can be up to at least 4 kb, if not larger.
The invention also relates to host cells which harbour a modified plasmid according to the invention, in particular a plasmid which comprises the sequence SEQ ID No. 1 and all or part of the sequence SEQ ID No. 2 into which a heterologous nucleotide sequence is inserted.
These host cells are, in particular, lactic acid bacteria, more especially belonging to the genera Leuconostoc, Lactobacillus and Pediococcus, preferably to the species Leuconostoc mesenteroides, Lactobacillus sake and Pediococcus acidilactici.